Ascorbic acid is present in high concentrations in the ocular tissues and aqueous humor of most diurnal animal species. Several lines of evidence support the concept that ocular ascorbic acid plays an important role in preventing tissue damage that otherwise could result from light, reactive oxygen species or other insults such as inflammation. In the course of scavenging reactive molecular species, ascorbic acid becomes partially or totally oxidized, resulting in formation of the ascorbate free radical (AFR) or dehydroascorbic acid (DHA), respectively. The present proposal addresses the hypothesis that specific enzymatic and transport mechanisms exist that recycle the toxic oxidative products to maintain a high ocular content of the useful reduced form of the molecule. The hypothesis will be tested that DHA is taken up into the cornea, lens, ciliary body - iris and retina by specific transport mechanisms and is subsequently reduced by intracellular DHA - reductase. Standard in vitro incubation conditions for each tissue will be selected on the basis of prior studies. Classic indications of transport phenomena will be sought, including saturation kinetics of substrate uptake, inhibition by structural analogs and dependence on cellular metabolic energy. The primary sites and properties of ocular DHA - reductase are of interest. Tissue samples are homogenized in buffer, fractionated by ammonium sulfate precipitation, desalted by dialysis and centrifuged one hour at 100,000 xg. The level of DHA - reductase following each step of the procedure is presented as the amount of 14C-DHA (10 uM) reduced during a 5-20 minute incubation. The enzyme's requirement for reducing equivalents optimum pH and characteristics of storage will be assessed. The techniques of affinity chromatography will be used to isolate and purify the enzyme. An effort will be made to determine if the enzyme is inducible. Similar types of studies will be performed on aqueous humor to determine whether this extracellular fluid that is lacking in superoxide dismutase and catalase, has the capacity to regenerate ascorbate. A second enzyme of interest is a soluble AFR - reductase; this activity would serve to limit DHA formation by reducing AFR before pairs of them disproportionate to form DHA and ascorbic acid. Recent evidence has suggested that errors of ascorbic acid metabolism or its redox system might be the cause of cataract formation Thus, the mechanisms by which ocular ascorbate is maintained in the reduced state assume considerable clinical importance.